The innate immune system performs the critical task of immediately protecting the host from infection and generating responses that alert and guide the actions of acquired immunity. Toll-like receptors (TLRs) are essential elements of innate immunity which upon direct recognition of microbial, fungal and viral components activate cellular events leading to inflammation, direct killing of the pathogen, and enhanced antigen presentation. Their essential role in inducing and regulating these events, is evidenced by their direct association with a variety of immune disorders ranging from sepsis, atherosclerosis, arthritis, asthma and autoimmunity. Among the ten member TLR family, TLR2 requires TLR1 or TLR6 to recognize, discriminate and initiate responses to a wide variety of rnicrobial components. Host responses are mediated by the two cytoplasmic proximal adaptor molecules known as MyD88 and TIRAP/MAL. The long term goal of this project is to define the mechanism by which this subfamily of TLRs recognize their agonists, initiate cellular responses, and coordinate these responses at the subcellular and molecular level. The specific aims are 1) To define the precise role of TLRs 1 and 6 in recognition of microbial and fungal components as well as the extracellular regions of the receptors involved, 2) To determine the intracellular trafficking and physical interactions among TLRs 1, 2 and 6 and the proximal adaptor molecules MyD88 and TIRAP/ MAL upon agonist-mediated cell activation, and 3) To define the role of proximal signaling events;on the observed cell surface colocalization of TLRs and other TLR-associated endocytic trafficking events. The specific function of TLRs, with respect to agonist discrimination as well as the structural basis of this discrimination, are assessed through the use of synthetic;agonists and receptor domain swapping experiments. The localization of TLRs and adaptors are assessed through direct microscopy including FRET, membrane fractionation, and pharmacalogic agents. The effect of dominant negative molecules, shRNA and pharmacalogic agents address the role of signaling events on intracellular TLR trafficking.